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Bovine Serum Albumin
Company: BSA) 50 mg/ml
Catalog#: 15561020
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The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein
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2020-08-05
[Abstract]  ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged ... [摘要]  [摘要]ATP酶是将ATP分解为ADP并释放无机磷（Pi）的酶。在这里，我们提供了一个详细的方案来测定重组AAA+-ATPase蛋白（一般对照非抑制-4[GCN4]）的ATPase活性，在360nm处测定无机磷的累积量。一般而言，底物2-氨基-6-巯基-7-甲基嘌呤核苷（甲基硫代鸟苷，鸟苷类似物：MESG）在Pi存在下通过嘌呤核苷磷酸化酶（PNP）酶转化为核糖1-磷酸和2-氨基-6-巯基-7-甲基嘌呤。测量了由于酶转化而导致的MESG底物和随后的转化产物在330nm处的最大吸收度的分光光度偏移。将gcn4his标记的重组蛋白在大肠杆菌BL21细胞中表达，并用Ni-NTA柱纯化。将纯化后的蛋白质用于溶液中Pi的定量测定或连续测定由于GCN4的atp酶活性而释放的Pi，GCN4是一种在许多真核生物中保存的AAA+-ATPase蛋白，在植物受到生物和非生物胁迫时调节气孔开度。

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